Abstract Cholesterol gallstone disease is one of the most prevalent digestive diseases and affects 12% of American adults, leading to a considerable financial and social burden in the USA. Discovered in 2001, apolipoprotein A5 (apoA5) is a new member of the apolipoprotein family and is synthesized exclusively in the liver. Although its plasma concentration is extremely low (114-258 ng/mL), humans and mice lacking functional apoA5 develop severe hypertriglyceridemia. We found recently that apoA5, a liver-specific protein, is also secreted into bile in mice and rats. This novel discovery greatly stimulates our curiosity to investigate whether apoA5 plays a previously unrecognized and critical role in biliary cholesterol homeostasis and gallstone formation. Our preliminary data show for the first time that: (i) apoA5 is secreted into bile and is associated solely with vesicles, but not micelles, in bile of wild-type mice. Hepatic expression and bile concentrations of apoA5 are significantly reduced in the lithogenic state. (ii) ApoA5 plays a pivotal role in cholesterol solubility in bile by changing the physical state of cholesterol carriers. (iii) The absence of apoA5 in bile disrupts biliary cholesterol homeostasis by promoting the aggregation and fusion of unilamellar vesicles to form unstable multilamellar vesicles, leading to rapid cholesterol crystallization. (iv) The lack of apoA5 in bile impairs gallbladder emptying and refilling, promoting the accumulation of excess mucin gel and the growth and agglomeration of solid cholesterol crystals into microlithiasis. (v) Overexpression of APOA5 in the liver reduces susceptibility to gallstones in human APOA5 transgenic mice, even fed a lithogenic diet. Based on these novel findings, we hypothesize that apoA5 plays a critical role in biliary cholesterol homeostasis, and its deficiency greatly enhances cholelithogenesis by promoting hepatic cholesterol hypersecretion, reducing cholesterol solubility in bile, and impairing gallbladder motility function. We further propose that there are both extracellular (in bile) and intracellular (in the liver and gallbladder) roles for apoA5 in increasing gallstone formation. To test the hypothesis, we will investigate the mechanisms underlying the lithogenic roles of apoA5 deficiency in disrupting cholesterol metabolism in the bile, liver and gallbladder, thereby enhancing cholelithogenesis. In addition, we will explore whether adeno-associated virus 2/8 (AAV2/8)-mediated gene transfer of the human APOA5 protects against gallstone formation by restoring normal hepatic and biliary cholesterol metabolism. After completing the proposed studies, our results will likely present a new view on how apoA5 regulates biliary cholesterol metabolism and will develop novel concepts to elucidate the critical roles of apoA5 in driving the initiation of supersaturated bile and cholesterol crystallization, two critical steps in the earliest stage of gallstone formation. We are confident that the successful completion of this project will provide novel insight into the mechanisms of elucidating the vital extracellular and intracellular roles of apoA5 in the regulation of hepatic and biliary cholesterol metabolism and the pathogenesis of gallstones.